3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Succinate dehydrogenase in Parkinson’s disease

Background

The prevalence of neurodegenerative disorders such as Parkinson’s disease (PD) is increased by age. Alleviation of their symptoms and protection of normal neurons against degeneration are the main aspects of the researches to establish novel therapeutic strategies. Many studies have shown that mitochondria as the most important organelles in the brain which show impairment in PD models. Succinate dehydrogenase (SDH) as a component of the oxidative phosphorylation system in mitochondria connects Krebs cycle to the electron transport chain. Dysfunction or inhibition of the SDH can trigger mitochondrial impairment and disruption in ATP generation. Excessive in lipid synthesis and induction of the excitotoxicity as inducers in PD are controlled by SDH activity directly and indirectly. On the other hand, mutation in subunits of the SDH correlates with the onset of neurodegenerative disorders. Therefore, SDH could behave as one of the main regulators in neuroprotection.

Objective

In this review we will consider contribution of the SDH and its related mechanisms in PD.

Methods

Pubmed search engine was used to find published studies from 1977 to 2016. “Succinate dehydrogenase”, “lipid and brain”, “mitochondria and Parkinson’s disease” were the main keywords for searching in the engine.

Results

Wide ranges of studies (59 articles) in neurodegenerative disorders especially Parkinson’s disease like genetics of the Parkinson’s disease, effects of the mutant SDH on cell activity and physiology and lipid alteration in neurodegenerative disorders have been used in this review.

Conclusion

Mitochondria as key organelles in the energy generation plays crucial roles in PD. ETC complex in this organelle consists four complexes which alteration in their activities cause ROS generation and ATP depletion. Most of complexes are encoded by mtDNA while complex II is the only part of the ETC which is encoded by nuclear genome. So, focusing on the SDH and related pathways which have important role in neuronal survival and SDH has a potential to further studies as a novel neuroprotective agent.

     

χ alcohol dehydrogenase isozymes of horse liver

Horse liver contains previously unrecognized isozymes of alcohol dehydrogenase. In contrast to the molecular forms identified up to now, under the conditions employed these variants migrate toward the anode on starch gel electrophoresis and were separated from the cathodic isozymes by DEAE-cellulose chromatography. They were then purified on agarose-hexane-AMP. Their physicochemical and compositional characteristics are similar to those x alcohol dehydrogenases from human liver. Like these and similar ones from simian liver, they retain most of their activity in the presence of10 mm 4-methylpyrazole, oxidize short-chain primary alcohols very poorly, and appear to prefer longer chain primary alcohols and ω-hydroxy fatty acids as substrates.     

The so-called "testis-specific aldehyde dehydrogenase" corresponds to type 2 retinaldehyde dehydrogenase in the mouse

The distribution pattern of "testis-specific aldehyde dehydrogenase" in mouse tissues was investigated. Because of the broad substrate specificity and the high degree of sequence identity of the large aldehyde dehydrogenase family a specific detection of single isoforms is not possible by histochemical means. Therefore, the technique of native isoelectric focusing was used. Thus, the expression of four to five banded "testis-specific aldehyde dehydrogenase" in the mouse testis was confirmed. However, the activity of this enzyme with the same pattern of multiplicity was found not only in the testis but also in the uterus and in embryonic tissues. At 9.5 and 10.5 days of embryonic development the enzyme activity was restricted to tissues of the embryonic trunk and absent in extracts from cranial tissues. The tissue distribution as well as substrate specificity and isoelectric points indicate that the "testis-specific aldehyde dehydrogenase" corresponds to mouse type 2 retinaldehyde dehydrogenase.     

The NAD-linked α-glycerophosphate dehydrogenase of trypanosomes

NAD-linked α-glycerophosphate dehydrogenase plays a key role in the α-glycerophosphate cycle of $\text{Trypanosoma brucei}$. The activity in cell lysates was ample for this role. The enzyme was activated by salts (e.g. MgCl₂ or NaCl); it had a broad pH-optimum for the reduction of dihydroxyacetone phosphate centred at pH 7.4, with an apparent K_m of 0.5 mM; and it was weakly bound to particulate components of cell lysates. The enzyme from $\text{T. vivax}$ was similar to that of $\text{T. brucei}$. These trypanosomal enzymes resemble that of the trypanosomatid $\text{Crithidia fasciculata}$, but are rather different from the enzymes of mammals, birds and insects.     

Inhibition of human and rat 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3β-HSD activity with IC₅₀ of 1.35 ± 0.05 and 1.77 ± 0.04 μM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 μM. All chemicals tested weakly inhibited human 3β-HSD activity with IC₅₀s over 250 μM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17β-HSD3 activity with IC₅₀s of 6.02 ± 1.02, 4.39 ± 0.46 and 127.60 ± 28.52 μM, respectively. The potencies for inhibition of 17β-HSD3 activity were determined to be PFOSK > PFOS > PFOA > PFHxSK = PFBSK for human 17β-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC₅₀s of PFOS(K) for inhibition of rat 17β-HSD3 activity was greater than 250 μM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3β-HSD and human 17β-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.     

Alanine dehydrogenase and its applications – A review

Alanine dehydrogenase (AlaDH) (E.C.1.4.1.1) is a microbial enzyme that catalyzes a reversible conversion of L-alanine to pyruvate. Inter-conversion of alanine and pyruvate by AlaDH is central to metabolism in microorganisms. Its oxidative deamination reaction produces pyruvate which plays a pivotal role in the generation of energy through the tricarboxylic acid cycle for sporulation in the microorganisms. Its reductive amination reaction provides a route for the incorporation of ammonia and produces L-alanine which is required for synthesis of the peptidoglycan layer, proteins, and other amino acids. Also, AlaDH helps in redox balancing as its deamination/amination reaction is linked to the reduction/oxidation of NAD⁺/NADH in microorganisms. AlaDH from a few microorganisms can also reduce glyoxylate into glycine (aminoacetate) in a nonreversible reaction. Both its oxidative and reductive reactions exhibit remarkable applications in the pharmaceutical, environmental, and food industries. The literature addressing the characteristics and applications of AlaDH from a wide range of microorganisms is summarized in the current review.     

Thermal stability of α-glycerophosphate dehydrogenase in Drosophila

Thermal stability of -glycerophosphate dehydrogenase-1 (-Gpdh-1) in nine Drosophila species was studied at pH's ranging from 6.4 to 8.5. This was done by measuring the changes in the activity of enzymes during the heat denaturation process. In addition to temperature, the rate of denaturation is highly dependent on the pH of the incubation buffer. The results of this study show that the thermal stability of enzyme molecules is different in different species. This holds true also in the species in which the enzymes have been found to be identical by other means. The differences between species of the Drosophila virilis group are discussed.This study was supported by funds from the National Research Council of Sciences of Finland.     

Mammalian alcohol dehydrogenase — Functional and structural implications

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1–ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system.     

β-Alanine suppresses heat inactivation of lactate dehydrogenase

β-Alanine exhibits neurotransmitter activity and is a component of the anti-glycation agent carnosine. We propose that β-alanine may have additional properties which may be of physiological significance. Interestingly, stress modulates the level of β-alanine, which regulates excitotoxicity responses and prevents neuronal cell death. We hypothesize that β-alanine's protective role may involve preservation of enzyme structure and function, suggesting that β-alanine may act as a chemical chaperone. We used light scattering, enzyme activity and intrinsic fluorescence to monitor heat-induced changes in lactate dehydrogenase (LDH) in the presence and absence of β-alanine. We observed that β-alanine suppressed heat-induced LDH inactivation, prevented LDH aggregation, ameliorated the decrease in intrinsic fluorescence and reactivated thermally denatured LDH. These observations support the hypothesis that β-alanine has chaperone-like activity and may play a cellular role in the preservation of enzyme function.     

Quantitative subunit hybridization of drosophilaα-Glycerophosphate dehydrogenase

Summary The dimeric enzyme,-Glycerophosphate dehydrogenase, was purified from eight Drosophila species by the method of Collier et al. (1976). The enzymes were inactivated at high pH and the conditions sufficient for reactivation were established. Electrophoretic patterns of reactivated-glycerophosphate dehydrogenases which were mixed following inactivation of two species' enzymes, demonstrate that high pH dissociates the enzyme into its constituent subunits and reactivation involves subunit reassociation. Twenty interspecific combinations of dissociated enzymes were allowed to reassociate, and the amounts of both heterospecific and homospecific enzyme activity and protein were determined by densitometry. In all 20 tests there were no differences between observed and expected heterospecific:homospecific enzyme ratios. These results are consistent with the very slow rate of evolution of this enzyme in the family Drosophilidae (Collier and MacIntyre, 1977).     

Robust NADH-regenerator: improved α-haloketone-resistant formate dehydrogenase

Formate dehydrogenases (FDH) are useful for the regeneration of NADH, which is required for asymmetric reduction by several dehydrogenases and reductases. FDHs have relatively low activity and are labile, especially to -haloketones, thus FDH cannot be applied to the industrial manufacture of optically active -haloalcohols. To stabilize a FDH from Mycobacterium vaccae (McFDH) against the -haloketone ethyl 4-chloroacetoacetate (ECAA), a set of cysteine-mutant enzymes was constructed. Sensitivity to ECAA of mutant C6S was similar to that of the wild-type enzyme, and mutants C249S and C355S showed little activity. In contrast, mutant C256S exhibited remarkable tolerance to ECAA. Surprisingly, mutant C146S was activated by several organic compounds such as ethyl acetate. An optimized mutant, C6A/C146S/C256V (McFDH-26), was obtained by combining several effective mutations. Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-ECHB] was synthesized from ECAA to 49.9 g/l with an optical purity of more than 99% e.e. using recombinant Escherichia coli cells coexpressing McFDH-26 and a carbonyl reductase (KaCR1) from Kluyveromyces aestuarii.     

DoesStreptomyces aureofaciens contain two alanine dehydrogenase isozymes ?

Summary Two NAD-dependent dehydrogenases exhibiting activity with L-alanine were detected in crude extracts ofStreptomyces aureofaciens. The dominant enzyme was found to be an alanine dehydrogenase with 6 or 4 subunits, each ofM, 45,000. The minor enzyme exhibited high activities with branched-chain amino acids, suggesting it is a valine dehydrogenase. The results demonstrate thatS. aureofaciens has only one alanine dehydrogenase, which is catalytically active in a six- or four-subunit structure.     

α-Ketoglutarate dehydrogenase: a mitochondrial redox sensor

α-Ketoglutarate dehydrogenase (KGDH), a key regulatory enzyme within the Krebs cycle, is sensitive to mitochondrial redox status. Treatment of mitochondria with H?O? results in reversible inhibition of KGDH due to glutathionylation of the cofactor, lipoic acid. Upon consumption of H?O?, glutathione is removed by glutaredoxin restoring KGDH activity. Glutathionylation appears to be enzymatically catalysed or require a unique microenvironment. This may represent an antioxidant response, diminishing the flow of electrons to the respiratory chain and protecting sulphydryl residues from oxidative damage. KGDH is, however, also susceptible to oxidative damage. 4-Hydroxy-2-nonenal (HNE), a lipid peroxidation product, reacts with lipoic acid resulting in enzyme inactivation. Evidence indicates that HNE modified lipoic acid is cleaved from KGDH, potentially the first step of a repair process. KGDH is therefore a likely redox sensor, reversibly altering metabolism to reduce oxidative damage and, under severe oxidative stress, acting as a sentinel of mitochondrial viability.     

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.     

Curcumin derivatives inhibit testicular 17β-hydroxysteroid dehydrogenase 3

Non-steroidal compounds that inhibit 17β-hydroxysteroid dehydrogenase isoform 3 (17β-HSD3), an enzyme catalyzing the final step in testosterone biosynthesis in Leydig cells, are under development for male contraceptive or treatment of androgen dependent diseases including prostate cancer. A series of curcumin analogues with more stable chemical structures were compared to curcumin as inhibitors of 17β-HSD3 in rat intact Leydig cells as well as rat and human testis microsomes.     

Kinetic mechanism of porcine testicular 17ß-hydroxysteroid dehydrogenase

A study on product inhibition of 17ß-hydroxysteroid dehydrogenase from porcine testes was carried out by measuring the initial velocities of NADPH formation using testosterone as the substrate steroid. Type of inhibition by NADPH against NADP⁺ was competitive in both saturated and unsaturated concentrations of testosterone. In the saturated concentration of NADP⁺, activity of the enzyme was not inhibited by NADPH against testosterone. In the unsaturated concentrations of NADP⁺, however, NADPH brought mixed type inhibition against testosterone. The similar modes of inhibition by the product steroid, androstenedione were observed in the saturated and unsaturated concentrations of NADP⁺ and testosterone.The fluorescence of NADPH was increased in the presence of the enzyme, and fluorometric titration indicated that 1 mol of NADPH was bound to 1 mol of the 17ß-hydroxysteroid dehydrogenase. Addition of testosterone to enzyme-NADPH complex reduced the intensity of fluorescence of NADPH, suggesting formation of testosterone-enzyme-NADPH complex as a ternary dead end complex.From the analyses of product inhibition and spectral changes of NADPH, the kinetic mechanism of the enzyme was revealed as rapid equilibrium random system with two dead end complexes which consisted of the two reduced reactants bound to the enzyme and the two oxidized ones bound to it.     

Chronic food restriction up-regulates 11-β-hydroxysteroid dehydrogenase

Corticosterone — product of 11-β-hydroxysteroid dehydrogenase type I (11βHSD1) stimulates adipocytes differentiation and activates lipogenic enzymes gene expression in white adipose tissue (WAT) of rats. The aim of the study was to examine the effect of chronic food restriction, often practised by obese individuals trying to lose body mass, on: a) 11βHSD1 gene expression, b) expression of genes associated with adipocyte differentiation (PPARg, SREBP-1, adiponectin), and c) expression of genes associated with lipogenesis in WAT of rats. Two-month old rats were divided into a control and a food restricted group obtaining 50% of food consumed by controls for 30 days. mRNA levels of studied genes in perirenal WAT were analysed by real-time PCR. 11βHSD1 and lipogenic enzymes activities were measured by radiometric conversion assay and by spectrophotometric assay respectively. Food restriction caused significant increase of 11βHSD1, PPARg, SREBP1, adiponectin and lipogenic enzymes mRNA levels in perirenal WAT. 11βHSD1 and some lipogenic enzymes activities were also increased by food restriction. The coordinated up-regulation of 11βHSD1, and genes associated with adipocyte differentiation and lipogenesis by food restriction suggests that such nutritional condition shifts WAT metabolism, that would permit this tissue to synthesize and accumulate triacylglycerols immediately after refeeding.